Apoptosis Induction And The Mechanisms Of Action Of Novel α, β-Unsaturated Carbonyl Compounds In Human Leukemia Cells

Theα,β-unsaturated carbonyl moiety existed in natural and synthetic compounds has been found to play an essential role in their antitumor activities.In this thesis,the structure and activity relationships in term of apoptosis induction were analyzed in two groups of compounds containing anα,β-unsaturated carbonyl group,ethacrynic acid derivatives and dimethylaminomethyl cyclopentanone derivatives.Our results revealed that the entity ofα,β-unsaturated carbonyl is required for apoptosis induction.Ethacrynic acid butyl-ester(EABE) and 2-(E)-pentylidene-5-dimethylaminomethyl cyclopentanone hydrochloride(WB852)have been found to be one of the most effective apoptosis inducers among each group of those compounds,respectively.Both EABE and WB852 have been used as a representative of each group for mechanism studies.EABE-induced apoptosis in leukemia cells correlated with the increased levels of reactive oxygen species(ROS),up-regulation of death receptor 5(DR5),the caspase activation,and the decreased levels of the mitochondrial membrane potential(MMP). Pretreatment with antioxidants,either N-acetylcysteine(NAC)or catalase(CAT),completely blocked EABE-induced apoptosis,H₂O₂ accumulation,and up-regulation of DR5 protein. Although WB852-induced apoptosis correlated with the ROS accumulation and MMP decrease, the levels of death receptors DR4,DR5 and Fas were not influenced by WB852 treatment. Unlike EABE,pretreatment with CAT did not block WB852-induced apoptosis.However, NAC counteracted the apoptosis induction of WB852.The difference of action between EABE and WB852 might be due to their ability of binding to a sulfhydryl group.Although both EABE and WB852 significantly depleted intracellular reduced glutathione(GSH)content, ESI-MS analyses revealed that WB852,but not EABE,nonenzymatically conjugated with GSH in a cell free system.Leukemia cells with high endogenous or overexpressed GSTP1-1 levels,which may catalyze the conjugation of EABE or WB852 with GSH,were less or insensitive to both agents-induced apoptosis.The synthetic conjugates of EA-GSH and WB852-GSH had lost or decreased apoptosis-induction abilities.WB852-induced apoptosis correlated with the activated caspase-8 that may probably resulted from the decreased levels of c-FLIP,one caspase-8 inhibitor.We conclude that EABE induces apoptosis in leukemia cells through a ROS-mediated pathway and WB852 induces apoptosis through a caspase-8-mediated pathway.GSTP1-1 and GSH play negative roles in EABE-and WB852-induced apoptosis.